CN109852591A - A kind of method that soya whey wastewater extracts lipoxygenase against pH gradient - Google Patents
A kind of method that soya whey wastewater extracts lipoxygenase against pH gradient Download PDFInfo
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Abstract
The invention discloses a kind of methods that soya whey wastewater extracts lipoxygenase against pH gradient, belong to food processing wastewater and utilize technical field.After the present invention pre-processes soya whey wastewater, lipoxygenase is extracted by inverse pH gradient method, foreigh protein removing is first removed at the pH far from lipoxygenase isoelectric point, and lipoxygenase is then precipitated at lipoxygenase isoelectric point again, obtains corps oxygenase in conjunction with ultrafiltration and gel chromatography.The present invention obtains the high lipoxygenase of industrial value using soya whey wastewater, and lipoxygenase recovery rate is high, with high purity, enzymatic activity is good.The method of the present invention is safe and efficient convenient for operating, at low cost, can be used for lipoxygenase in industrial soya whey wastewater and develops and uses, to realize that resource utilization utilizes, has significant economic benefit and social benefit.
Description
Technical field
The present invention relates to food processing wastewaters to utilize technical field, in particular to a kind of soya whey wastewater is mentioned against pH gradient
The method for taking lipoxygenase.
Background technique
Soybean is a kind of protein content important crops more abundant, using it as the soybean protein isolate of Raw material processing
It is that a kind of purity of protein is high, the Refined Soybean protein product with deep shaping property, can be used as intermediate raw material, to be added to food raw
It is widely used in production.
Soybean protein isolate can use alkali extraction-acid precipitation, ultrafiltration concentration method, inverse micelle abstraction method, reversed-phased high performace liquid chromatographic
The methods of processed, wherein alkali extraction-acid precipitation is that current production soybean protein isolate is more mature, widely applied technique side
Method.But a large amount of soya whey wastewater can be generated sinking while technique produces soybean protein isolate using alkali soluble acid, and is given up
The substances such as albumen rich in, carbohydrate in water, easily result in waste of resources and environmental pollution.
Soybean protein isolate is widely used in food industry production, and annual demand is but every at 600,000 tons or so
The waste water that 1 ton of SPI will generate 40 tons or so is produced, the yield of waste water is very big.Currently, for soybean protein isolate processing waste water
Main methods be directly to be carried out multi-stage biological processing, using anaerobic and aerobic method reduce waste water in COD and
BOD, to reach national wastewater discharge standard, the processing cost is high, and a large amount of active material contained in waste water is not also closed
Reason utilizes.Therefore, benefit can be recycled by recycling the equal high value added products in soya whey wastewater using modern separation technology
With efficient resource, sewage is purified, preferable economic benefit and social benefit are generated.
In recent years, in order to reduce discharge of wastewater and environmental pollution, many scholars start to be dedicated to function in soya whey wastewater
The separation and Extraction, such as Jiang etc. of energy property substance recycle soybean using two-part foam separating technology from soya whey wastewater
Lactalbumin；Ling Xiuju etc. extracts the oligosaccharide in soya whey wastewater using membrane technology, and forms soy oligosaccharides sugar product.
However, the research about lipoxygenase separation and Extraction in soya whey wastewater has no research report.
Lipoxygenase (LOX), also known as lipoxidase, lipoxygenase belong to oxidoreducing enzyme, are a kind of containing non-heme
The protein of iron, single-minded can be catalyzed has suitable, the polyunsaturated fatty acid of suitable 4- alkene structure, passes through intramolecular oxygenation, formation tool
There is the hydro-oxidation derivative of conjugated double bond.Lipoxygenase (LOX) can lead to garden stuff processing product generate bad flavor, grease and
Deterioration etc. occurs for oil-containing food color during storage and processing.But lipoxygenase (LOX) adds as green food
Add agent, in wheat flour, the pigment in oxidable flour to be allowed to fade, brightens Flour product.Lipoxygenase (LOX) can
To apply to plant disease-resistant, in pest-resistant and shock reaction, the relationship of lipoxygenase (LOX) and plant disease-resistant wheat, tobacco,
It is all had been reported that on the crops such as rice, cotton.Lipoxygenase (LOX) industrially can be used for the work of dyestuff, coating, detergent etc.
Industry metaplasia produces, and is alternatively arranged as the intermediate of pharmaceutical synthesis.
Although LOX has in the fields such as food, chemical industry and has been widely used, directly mentioned from the raw material such as soybean
It takes, cost is too high, and there are no works to become the enzyme preparation that industry metaplasia produces；And LOX is recombinated by genetic engineering bacterium high efficient expression and is deposited
In problems such as safeties, so that it be made to be difficult to biggish breakthrough in terms of large-scale production and application.As it can be seen that finding suitable
The method for closing the acquisition lipoxygenase of industrial application is the problem of current urgent need to resolve.
It is found in researcher's early-stage study, a large amount of lipoxygenase is contained in SPI processing waste water, China is in the world
The maximum every annual meeting of SPI producing country generates a large amount of SPI processing waste water, in consideration of it, to rouge in soybean protein isolate processing waste water
SPI processing waste water is carried out resource utilization by fat oxygenase separation and Extraction, will generate huge economic and social value, tool
There is highly important realistic meaning.
Summary of the invention
In order to make up for the deficiencies of the prior art, the present invention provides a kind of soya whey wastewaters extracts fatty oxygen against pH gradient
The method of synthase.
The technical solution of the present invention is as follows:
A kind of method that soya whey wastewater extracts lipoxygenase against pH gradient, comprising steps of
1) soya whey wastewater pre-processes
The impurity that soya whey wastewater is carried out to centrifugation removal bulky grain retains supernatant and pre-processes as soya whey wastewater
Liquid；
2) foreign protein in soya whey wastewater pretreatment fluid is removed
The pH of soya whey wastewater pretreatment fluid is adjusted to 7.8 ~ 8.2, is centrifuged off precipitating；
3) lipoxygenase is extracted
The pH value that step 2 is centrifuged resulting supernatant is adjusted to 5.7 ~ 6.0, centrifugation removal supernatant, gained sediment is steamed
Distilled water dissolution, as lipoxygenase crude enzyme liquid.
After the present invention pre-processes soya whey wastewater, lipoxygenase is extracted by inverse pH gradient method, first far from rouge
Foreigh protein removing is removed under the pH of fat oxygenase isoelectric point, and lipoxygenase is then precipitated at lipoxygenase isoelectric point again.
Preferably, the method that the soya whey wastewater extracts lipoxygenase against pH gradient, further includes step
4): lipoxygenase crude enzyme liquid utilizes the method preliminary purification being concentrated by ultrafiltration.
Preferably, the method that the soya whey wastewater extracts lipoxygenase against pH gradient, further includes step
5): enzyme solution obtained by step 4) is further purified using gel filtration chromatography, obtains pure lipoxygenase.
Preferably, in step 4), using the modified poly (ether-sulfone) ultrafiltration membrane of 30KDa size, in pressure 200 ~ 300
8 ~ 10 times of ultrafiltration concentrations are carried out to lipoxygenase crude enzyme liquid under KPa.
Preferably, it in step 4), is concentrated by ultrafiltration in such a way that ultrafiltration system is using double-filtration
Preferably, in step 5), gel filtration chromatography utilizes the Sephadex G-75 gel column of 2-70KDa, with
The phosphate buffer of pH7.8,0.02mol/L balances 2 ~ 3 column volumes, loading after balance, with pH7.8,0.02mol/L's
Phosphate buffer is elution；Eluent flow rate is 0.5mL/min.
Preferably, in step 2, the pH of soya whey wastewater pretreatment fluid is adjusted to 8.0.
Preferably, in step 3), the pH value of supernatant is adjusted to 6.0.
Preferably, in step 2 and step 3), the pH of sodium hydroxide solution regulation system is utilized.
Preferably, in step 1), 8 ~ 20min is centrifuged with the revolving speed of 3000 ~ 15000r/min.
The invention has the benefit that
1, the present invention provides a kind of method that soya whey wastewater extracts lipoxygenase against pH gradient, the present invention is first to soybean
Then whey wastewater pretreatment adjusts pH to far from lipoxygenase isoelectric point, first cleans, then adjusts pH to lipoxygenase
Isoelectric point obtains the lipoxygenase of high enzyme activity and the high enzyme activity rate of recovery, and the enzyme activity rate of recovery is 73.54%, and specific enzyme activity is up to
10661.32U/mg。
2, the present invention by isoelectric precipitation obtain lipoxygenase be further concentrated by ultrafiltration, gel filtration chromatography
Purifying obtains the very high lipoxygenase of purity, and final LOX purity is 90.43%, and specific enzyme activity reaches 25546.08U/mg, enzyme activity
The rate of recovery is 40.29%, and purification improves 25.44 times.
3, the present invention obtains the high lipoxygenase of industrial value using soya whey wastewater, and lipoxygenase is extracted
Rate is high, with high purity, enzymatic activity is good.
4, the method for the present invention is safe and efficient convenient for operating, at low cost, can be used for fatty in industrial soya whey wastewater
Oxygenase development and utilization have significant economic benefit and social benefit to realize that resource utilization utilizes.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this
Some embodiments of invention without any creative labor, may be used also for those of ordinary skill in the art
To obtain other drawings based on these drawings.
Fig. 1 is protein content determination canonical plotting；
Fig. 2 is the rate of deposition and specific enzyme activity figure of lipoxygenase (LOX) under each pH；
Fig. 3 is pH gradient precipitating proteins electrophoretogram；
Fig. 4 is precipitating and LOX specific enzyme activity figure in supernatant under each PEG6000 concentration；
Fig. 5 is influence situation map of the Sodium Polyacrylate additional amount to lipoxygenase specific enzyme activity；
Fig. 6 is influence situation map of the ultrafiltration retaining molecular weight to the LOX enzyme activity rate of recovery and ultrafiltration time；
Fig. 7 is influence situation map of the ultrafiltration pressure to the LOX enzyme activity rate of recovery；
Fig. 8 is that multiple is concentrated by ultrafiltration to the unit enzyme activity of LOX and the influence situation map of the enzyme activity rate of recovery；
Fig. 9 is influence situation map of the ultrafiltration mode to LOX unit enzyme activity and the enzyme activity rate of recovery；
Figure 10 is that Sephadex G-75 chromatographs map；
Figure 11 is that the SDS-PAGE of LOX purification process analyzes result.
Specific embodiment
One, lipoxygenase enzyme activity determination method in present embodiment:
Enzyme activity definition: at 25 DEG C, under the conditions of pH9.0, using linoleic acid as substrate, at 234nm, reaction system 1min catalysis is generated
The enzyme concentration of 1umol product is defined as an enzyme-activity unit.
Substrate preparation: the polysorbas20 of 0.25mL is dispersed in the 0.2mol/L of 10mL, the borate buffer solution that pH is 9.0
In, the linoleic acid of 0.27mL is constantly shaken and be added dropwise, it is uniformly mixed, is then added dropwise into system certain density
NaOH solution clarifies system, and regulation system pH to 9.0, finally uses 0.2mol/L, and the borate buffer solution of pH9.0 is settled to
500mL is stored at 4 DEG C as substrate solution, spare.
Determination of activity: 0.2mL sample solution is added in 1.5mL substrate solution, the isothermal reaction at 25 DEG C after mixing
After 3min, 5mL dehydrated alcohol is added and terminates reaction, adding 5mL distilled water clarifies reaction system, measures and inhales at 234nm
Luminosity.Blank sample is that 5mL dehydrated alcohol is first added in 1.5mL substrate solution, and 0.2mL sample solution is being added, permanent at 25 DEG C
After warm 3min, 5mL distilled water is added.
The above enzyme activity determination method refers to following two document:
1, influence [D] Southern Yangtze University of the Wang Ren super-pressure to soybean lipoxygenase, nutrition inhibiting factor and property of protein,
2008.
2、 Catod L, Halmos A L, Small D M. Measurement of lipoxygenase in
Australian white wheat flour: the effect of lipoxygenase on the quality
properties of white salted noodles[J]. Journal of the Science of Food and
Agriculture, 2006, 86(11): 1670-1678. DOI: 10.1002/jsfa.2539.
Two, Various Methods for Determing Different Proteins in present embodiment:
Using the content of protein in the method measurement sample of Bradford, takes 6 clean tubes and mark pipe number, wherein one
1.0ml distilled water is added and does blank, 5 concentration for being separately added into different volumes are 0.1mg/ml bovine serum albumin standard liquid,
Replenish water to 1.0ml.Then 5.0ml Coomassie brilliant G-250 reagent is added in every test tube, shakes up and places 5min, it is ultraviolet-can
See measurement light absorption value at spectrophotometer 595nm.Using A595 light absorption value as ordinate, the concentration (mg/mL) of bovine serum albumin
Standard curve is drawn for abscissa, as shown in Figure 1.
Three, SDS-PAGE analysis experiment in present embodiment
SDS-PAGE analysis is carried out to LOX sample solution with reference to Laemmli method, sample is with 2Loading buffer carries out molten
Solution, and 3min is heated in boiling water bath, 3min is centrifuged with 8000r/min.Resolving gel concentration is 15%, and concentration gum concentration is 5%, electricity
Pole buffer is pH8.3 Tris-Gly buffer, and separation gel buffer liquid is 1.5mol/L pH8.8 Tris-HCl buffer,
Concentration glue buffer is 1.0mol/L pH6.8 Tris-HCl buffer, at room temperature electrophoresis 120min.With examining after electrophoresis
Mas bright blue R-250 coloring agent carries out dyeing 8h, and decoloration for 24 hours, then carries out photographic analysis.And according to protein content in sample
Linearly relevant principle carries out protein precipitation rate and purity analysis to PAGE gel electrophoretic band gray value, using solidifying
Glue imaging system acquires electrophoretogram, gray value analysis is carried out to each electrophoretic band with Image J software, based on formula (1) and (2)
Calculate protein precipitation rate (PR) and purity (PU) in each sample.
Wherein, PGproRepresent testing protein gray value in precipitating；EGproRepresent testing protein gray scale in soya whey wastewater
Value；Np and Ne respectively represents precipitating and soya whey wastewater extension rate；SGproTesting protein gray value in representative sample；
SGallRepresent the total gray value of all proteins in precipitating.
Four, lipoxygenase research is precipitated along pH gradient
The equal point of LOX takes each 6 parts of a certain amount of wastewater treatment liquid between pH5.7-6.0, wherein 1 part as a control group,
Remaining 5 parts are experimental group, are adjusted to pH5.0, pH6.0, pH7.0, pH8.0, pH9.0 respectively with the NaOH of 1mol/L, are stood in 4 DEG C
It is centrifuged 10min after 5h in centrifuge, collects supernatant and precipitating, wherein in precipitating and being dissolved and be adjusted to a certain amount of distilled water
Property, it is centrifuged 10min, supernatant is taken and measures the enzyme activity and protein content of wherein LOX, it is real to carry out analysis using SDS-PAGE
It tests, and measures rate of deposition of the LOX in precipitating, as a result as shown in Figure 2.
As shown in Figure 2, when soya whey wastewater pH being adjusted to 6.0, the specific enzyme activity of LOX and rate of deposition are above it in precipitating
Specific enzyme activity and rate of deposition under the conditions of its pH in precipitating, and specific enzyme activity is lower than the ratio in supernatant under the conditions of other pH in supernatant
Enzyme activity, the reason is that the isoelectric point due to LOX is located near pH6.0, when pH value of solution is 6.0, the ratio enzyme of LOX in precipitating
Living is 2816.56U/mg, and purification improves 2.80 times, and LOX rate of deposition is up to 76.82%.And when wastewater treatment liquid pH is adjusted to
When 8.0, the specific enzyme activity of LOX specific enzyme activity and rate of deposition lower than other pH conditions LOX and rate of deposition in precipitating, and LOX in supernatant
Specific enzyme activity be higher than the specific enzyme activity under the conditions of other pH in supernatant, this is because wastewater treatment liquid can sink under the conditions of pH8.0
Form sediment basic protein more out, and LOX is remained substantially in supernatant, therefore when pH value of solution is 8.0, LOX in supernatant
Specific enzyme activity is higher, and when pH value of solution is 6.0, the specific enzyme activity of LOX and rate of deposition are higher in precipitating.
Upper experimental result carries out inverse pH gradient experiment accordingly, and soya whey wastewater pH is first adjusted to separate LOX isoelectric point, with
The foreign protein of isoelectric point at this ph is removed, then centrifugation gained supernatant is adjusted at LOX isoelectric pH.
Embodiment 1
A kind of method that soya whey wastewater extracts lipoxygenase against pH gradient, comprising steps of
1) soya whey wastewater pre-processes
1000mL soya whey wastewater is taken, in 12000r/min, 10min is centrifuged, it is pre- as soya whey wastewater to retain supernatant
Treatment fluid, in case used in experiment；
2) foreign protein in soya whey wastewater pretreatment fluid is removed
The pH of soya whey wastewater pretreatment fluid is adjusted to 8.0 using sodium hydroxide solution, is centrifuged off precipitating；
3) lipoxygenase is extracted
The pH value that step 2 is centrifuged resulting supernatant is adjusted to 6.0 using sodium hydroxide solution, centrifugation removes supernatant, institute
It obtains sediment to be dissolved with distilled water, and adjusts pH to neutrality, as lipoxygenase crude enzyme liquid, measure enzyme activity and egg in crude enzyme liquid
White matter content, and analysis measurement is carried out to lipoxygenase rate of deposition and purity in conjunction with SDS-PAGE, as a result such as table 1 and Fig. 3 institute
Show.
Table 1 precipitates LOX parameter list against pH gradient
According under each pH precipitate soya whey wastewater in LOX result of study it is found that under the conditions of pH6.0 in soya whey wastewater
The sedimentation effect of LOX is preferable, and foreign protein in soya whey wastewater can be preferably removed under the conditions of pH8.0.Therefore, this experiment is first
Soya whey wastewater is adjusted at pH8.0, a part of foreign protein is removed, then gained supernatant is adjusted at pH6.0, precipitates mesh
Albumen, acquired results are shown in Fig. 3 and table 2.As seen from the figure, foreign protein contained by band 6 significantly reduces in Fig. 3, and in band
Protein molecular weight has marked difference, is conducive to further isolate and purify.And as shown in Table 1, LOX's is pure in pH8.0 supernatant
Changing multiple is 3.10, and the enzyme activity rate of recovery illustrates that this process plays certain purification effect to LOX up to 97.32%, and can be preferably
Holding LOX enzyme activity.When pH is adjusted to 6.0, the purification of LOX improves 10.62 times in precipitating, and specific enzyme activity is up to
10661.32U/mg, and the LOX enzyme activity rate of recovery and purifying are respectively 73.54% and 39.55%, it is seen that inverse pH gradient precipitation process pair
LOX plays preferable sedimentation effect.
Comparative example 1
The method of PEG6000 precipitating lipoxygenase
Bibliography: Yu Wuying, Zhao Jinmei, Xiao Qinghu wait the Xihua research [J] of polyethylene glycol precipitation Porcine trypsin
College journal (natural science edition), 2011,30 (5): 96-99.
It is added the 50% of different quality into a certain amount of soya whey wastewater pretreatment fluid respectively under the conditions of 4 DEG C
PEG6000 solution so that the mass fraction of PEG6000 is respectively 5%, 10%, 15%, 20%, 25%, 30% in solution, and stirs equal
Even 6h static at 4 DEG C is centrifuged 10min, gained after PEG6000 is sufficiently combined with protein under the conditions of 12000r/min
Precipitating is dissolved with 0.2mol/L pH8.0 borate buffer solution, measures LOX enzyme activity and protein content in solution, is used
SDS-PAGE carries out experimental analysis, and measures the rate of deposition of LOX in precipitating.
The result of study of LOX is as shown in Figure 4 in PEG6000 precipitation and separation soya whey wastewater.As shown in Figure 4, PEG6000
Concentration difference is different to the sedimentation effect of LOX, and when PEG6000 concentration is in 10% or less, LOX specific enzyme activity is equal in precipitating
Higher than the specific enzyme activity of supernatant and soya whey wastewater, when PEG6000 concentration is 10%, LOX purification and rate of deposition are most
Height, respectively 1.70 and 32.83%, illustrate that PEG6000 precipitation and separation technology has certain sink to the LOX in soya whey wastewater
Shallow lake effect.When the concentration of PEG6000 is higher than 10%, LOX specific enzyme activity is below LOX specific enzyme activity in supernatant in precipitating, and LOX is heavy
Shallow lake rate is on a declining curve, and LOX specific enzyme activity is not significantly increased in supernatant, so when PEG6000 concentration is higher than 10%,
It is poor to the sedimentation effect of LOX in soya whey wastewater.And studies have shown that: PEG molecule has extensibility in aqueous solution,
It is difficult to remove the PEG in protein solution, therefore PEG- protein conjugate purifying difficulty is big, purpose product must be measured lower.
Therefore, LOX rate of deposition and purification are integrated it can be concluded that isoelectric precipitation technology is to LOX in soya whey wastewater
Separating effect it is preferable.
Comparative example 2
Bibliography: Du Licheng, Yang Kuihua extract technical study [J] food and the fermentation section of lysozyme from egg shell
Skill, 2005,41 (2): 22-25.
6 parts of 20mL soya whey wastewaters are taken respectively, sequentially add 2mL, 3mL, 4mL, the polyacrylic acid of 5mL, 6mL 0.5%
Sodium stands flocculation sedimentation and is centrifugated precipitating and supernatant afterwards for 24 hours after mixing.Add into LOX- Sodium Polyacrylate precipitating
Enter a small amount of distilled water, and 0.5mol/L Na is added dropwise2CO3Sediment is dissolved, 5%CaCl is then added2It dissociates LOX and gathers
Sodium acrylate is finally centrifugated in centrifuge up to no Precipitation, and gained supernatant is used for enzyme activity determination and protein
Assay analyzes LOX rate of deposition in conjunction with SDS-PAGE.
Sodium Polyacrylate contains a large amount of carboxyl, can be formed by the protein of electrostatic interaction junction belt counter ion
Polyelectrolytes-protein complex precipitating, and its dosage is less, can be re-dissolved by water, therefore preferable holding
Protein active.Fig. 5 is using the result of study of LOX in 0.5% sodium polyacrylate solution precipitating soya whey wastewater, by Fig. 5
It is found that gained LOX specific enzyme activity is above the specific enzyme activity of LOX in supernatant in Sodium Polyacrylate-LOX complex precipitate, wherein LOX
Purification and rate of deposition highest are respectively up to 1.89 and 40.82%, it can be seen that, Sodium Polyacrylate is in soya whey wastewater
LOX has certain sedimentation effect, but the intermediate processing extraction effect is undesirable.
Embodiment 2
A kind of method that soya whey wastewater extracts lipoxygenase against pH gradient, comprising steps of
1) soya whey wastewater pre-processes
1000mL soya whey wastewater is taken, is centrifuged 10min in 12000r/min, retains supernatant and locates in advance as soya whey wastewater
Liquid is managed, in case used in experiment；
2) foreign protein in soya whey wastewater pretreatment fluid is removed
The pH of soya whey wastewater pretreatment fluid is adjusted to 8.0 using sodium hydroxide solution, is centrifuged off precipitating；
3) lipoxygenase is extracted
The pH value that step 2 is centrifuged resulting supernatant is adjusted to 6.0 using sodium hydroxide solution, centrifugation removes supernatant, institute
It obtains sediment to be dissolved with distilled water, and adjusts pH to neutrality, as lipoxygenase crude enzyme liquid；
4) lipoxygenase crude enzyme liquid utilizes the Methods For Purification being concentrated by ultrafiltration
Using ultrafiltration system and the modified poly (ether-sulfone) ultrafiltration membrane of 30KDa size, two-stage ultrafiltration mode is selected, in 200 KPa of pressure
Under to lipoxygenase crude enzyme liquid carry out 10 times ultrafiltration concentration.
In order to further increase the purity of LOX extracting solution, with ultrafiltration system and modified poly (ether-sulfone) ultrafiltration membrane, LOX is extracted
Liquid carries out that optimization experiment is concentrated by ultrafiltration, to probe into optimal ultra-filtration conditions for industrialized production LOX.
Optimization experiment is concentrated by ultrafiltration by carrying out using ultrafiltration system and modified poly (ether-sulfone) ultrafiltration membrane in the acquisition of conditions above.
In order to ensure LOX is retained completely, the higher enzyme activity rate of recovery is obtained, according to the molecular size range of LOX and ultrafiltration membrane Bao Xuan
With principle, answering selective retention molecular weight is the ultrafiltration membrane packet of LOX molecular weight one third to 1/6th, to probe into ultrafiltration membrane
Influence of the factors such as molecular cut off, operating pressure, ultrafiltration mode and cycles of concentration to ultra-filtration process.
The optimization of 2.1 ultrafiltration retaining molecular weights
Influence of the ultrafiltration membrane of different molecular weight cut off to the enzyme activity rate of recovery of LOX is different, according to LOX molecular size range and
The selection principle of ultrafiltration membrane packet, and the spare external member of comprehensive slipstream film packet, selective retention molecular weight is 10KDa, 30KDa respectively
And the ultrafiltration membrane of 50KDa carries out ultrafiltration concentration processing to extracting solution obtained by isoelectric point fractional precipitation.In ultra-filtration process, operation pressure
Power is 200KPa, carries out 6 times of concentrations to precipitating extracting solution using level-one ultrafiltration mode, different to probe into ultrafiltration retaining molecular weight
Influence to the LOX enzyme activity rate of recovery and ultrafiltration time, as a result as shown in Figure 6.
As seen from Figure 6, with the increase of ultrafiltration retaining molecular weight, the enzyme activity rate of recovery is successively reduced, and ultrafiltration is dense
Time used in identical multiple that contracts successively is reduced.When ultrafiltration retaining molecular weight is 10KDa, the enzyme activity rate of recovery is up to
70.84%, but the ultrafiltration time used is most.When ultrafiltration retaining molecular weight is 30KDa, the enzyme activity rate of recovery is 69.51%, institute's used time
Between be 196min, compared with the ultrafiltration membrane of 10KDa, the time greatly reduces.And ultrafiltration retaining molecular weight be 50KDa when LOX enzyme
The rate of recovery living is minimum, and the time used is also minimum.This is because ultrafiltration retaining molecular weight size can influence the membrane flux of ultrafiltration membrane,
And membrane flux is an important indicator for characterizing ultrafiltration membrane efficiency, i.e. ultrafiltration retaining molecular weight is bigger, and membrane flux is higher, ultrafiltration
Time is then shorter, and ultrafiltration efficiency is higher.But to ensure that LOX is retained completely, the higher enzyme activity rate of recovery is obtained, in ultra filtration
Ultrafiltration retaining molecular weight should be selected as the one third of LOX molecular weight to 1/6th, so that ultrafiltration membrane retains molecule in journey
Amount is lower than the molecular weight of molecule to be retained, and is higher than the molecular weight being intended to through molecule.Therefore, molecular cut off is the super of 50KDa
Filter membrane is poor to LOX rejection effect, and the ultrafiltration membrane that molecular cut off is 10KDa is best to the rejection effect of LOX, but institute's used time
Between it is longer, and in the industrial production, not only consider the LOX enzyme activity rate of recovery, also to consider the ultrafiltration time, therefore comprehensively consider
The LOX enzyme activity rate of recovery and ultrafiltration time select the ultrafiltration membrane that molecular cut off is 30KDa to carry out hyperfiltration treatment.
The optimization of 2.2 ultrafiltration pressure
In soya whey wastewater in addition to containing most lactalbumin, also contain the polysaccharide of some small molecules, such as soy oligosaccharides
It is increasing with the increase of ultrafiltration multiple to make the viscosity of soya whey wastewater for sugar, and operating pressure can also produce LOX
Raw certain loss, it is thus determined that suitable operating pressure can preferably keep the LOX enzyme activity rate of recovery.Cycles of concentration is 6 times, is surpassed
Membrane retention molecular weight is 30KDa, with probe into be concentrated by ultrafiltration during more appropriate operating pressure and time, result of study is such as
Shown in Fig. 7.
Figure such as 7 it is found that with operating pressure increase, the enzyme activity rate of recovery and ultrafiltration time successively reduces, when operation is pressed
When power is respectively 100KPa and 200KPa, the LOX enzyme activity rate of recovery is respectively 69.51% and 68.92%, and the ultrafiltration time used is respectively
196min and 161min, it can be seen that, it is very nearly the same in two operating pressure LOX enzyme activity rate of recovery, but the ultrafiltration time difference used
It is not more.When operating pressure is 300KPa, the LOX enzyme activity rate of recovery and ultrafiltration time are respectively 62.08% and 144min, and are pressed
The bigger LOX enzyme activity rate of recovery of power and ultrafiltration time used are smaller.This is because operating pressure is excessive in ultra-filtration process, ultrafiltration
Film surface is easy to produce concentration polarization phenomenon, in some instances it may even be possible to gel layer is formed, so that certain components in material are in fenestra
Deposition causes fenestra to reduce or blocking, and operating pressure is excessive easily causes film bags burst.Therefore in ultra-filtration process,
To improve UF membrane efficiency, Pollution of Ultrafiltration Membrane is reduced, is generally controlled operating pressure in 300KPa hereinafter, therefore operating pressure selection
200Kpa。
The optimization of 2.3 cycles of concentration
Influence of the different ultrafiltration multiples to LOX unit enzyme activity and the enzyme activity rate of recovery is also different, influences result such as Fig. 8 institute
Show.
As can be seen from Figure 8, with the increase that multiple is concentrated by ultrafiltration, LOX unit enzyme activity is successively increased, and enzyme activity recycles
Rate is gradually reduced.When being concentrated by ultrafiltration to 10 times, the unit enzyme activity and the enzyme activity rate of recovery of LOX be respectively 17946.88U/mL and
66.23%, and since the concentrate needs after ultrafiltration are further purified, ultrafiltration concentration multiple is bigger, gained concentrate
Volume it is smaller, and then greatly reduce the workload of subsequent purification process.
The optimization of 2.4 ultrafiltration modes
According to the above optimization experimental result, in ultra-filtration process, operating pressure 200KPa, and select the ultrafiltration membrane pair of 30KDa
Extracting solution obtained by isoelectric point fractional precipitation carries out 10 times of concentrations, and the selection of ultrafiltration mode can also be to the LOX enzyme activity rate of recovery and list
Position enzyme activity generates certain influence.
Level-one ultrafiltration and two-stage ultrafiltration mode is respectively adopted, concentration is carried out to isoelectric point fractional precipitation extracting solution, with true
Fixed more appropriate ultrafiltration mode, as a result as shown in Figure 9.
According to Fig. 9 as can be seen that LOX unit enzyme activity obtained by level-one ultrafiltration and the enzyme activity rate of recovery are respectively 17946.88U/mL
With 66.23%, and two-stage ultrafiltration is then that will precipitate extracting solution to be divided into equal two parts, and mix after being concentrated 3 times respectively
4 times of concentration, the final gained LOX enzyme activity rate of recovery and unit enzyme activity are respectively 19713.092U/mL and 71.08%.It is super according to two kinds
The final acquired results of filter mode select two-stage ultrafiltration mode to carry out sample treatment.
Under conditions of operating pressure is 200KPa, use molecular cut off for the ultrafiltration membrane of 30KDa and two-stage ultrafiltration side
Formula carries out 10 times of concentrations to extracting solution obtained by isoelectric point fractional precipitation, and the unit enzymatic activity of gained concentrate is 19713.092U/
ML, purification improves 16.79 times, and LOX purity and the enzyme activity rate of recovery are respectively up to 51.85% and 71.08%, it can be seen that,
Extracting solution obtained by processing isoelectric point fractional precipitation is advanced optimized using ultrafiltration system, is greatly improved the pure of LOX in extract liquor
Degree.
Embodiment 3
A kind of method that soya whey wastewater extracts lipoxygenase against pH gradient, comprising steps of
1) soya whey wastewater pre-processes
1000mL soya whey wastewater is taken, is centrifuged 10min in 12000r/min, retains supernatant and locates in advance as soya whey wastewater
Liquid is managed, in case used in experiment；
2) foreign protein in soya whey wastewater pretreatment fluid is removed
The pH of soya whey wastewater pretreatment fluid is adjusted to 8.0 using sodium hydroxide solution, is centrifuged off precipitating；
3) lipoxygenase is extracted
The pH value that step 2 is centrifuged resulting supernatant is adjusted to 6.0 using sodium hydroxide solution, centrifugation removes supernatant, institute
It obtains sediment to be dissolved with distilled water, and adjusts pH to neutrality, as lipoxygenase crude enzyme liquid；
4) lipoxygenase crude enzyme liquid utilizes the method preliminary purification being concentrated by ultrafiltration
Using ultrafiltration system and the modified poly (ether-sulfone) ultrafiltration membrane of 30KDa size, two-stage ultrafiltration mode is selected, in 200 KPa of pressure
10 times of ultrafiltration concentrations are carried out to lipoxygenase crude enzyme liquid.
5) enzyme solution obtained by step 4) is further purified using gel filtration chromatography, obtains pure lipoxygenase；Gel
Filtration Chromatography utilizes the Sephadex G-75 gel column of 2-70KDa, and with pH7.8, the phosphate buffer of 0.02mol/L is balanced
2 ~ 3 column volumes, loading after balance, with pH7.8, the phosphate buffer of 0.02mol/L is elution；Elute liquid stream
Speed is 0.5mL/min；Automatic collection sample merges appearance part, surveys LOX enzyme activity and protein content in its solution.
LOX crude enzyme liquid is further purified in selection gel filtration chromatography, and the results are shown in Figure 10 for elution.It can be with by Figure 10
Find out, by Sephadex G-75 gel chromatography, obtains 5 protein peaks, and enzyme activity is carried out to sample collected by each peak
Measurement, and be concentrated merging with the sample of LOX enzyme activity.
In order to further verify the purification result of LOX in soya whey wastewater, the sample of each purification phase is carried out
SDS-PAGE analysis, acquired results are as shown in table 2 and Figure 11.As can see from Figure 11 containing more in soya whey wastewater
Protein band, the molecular weight protein band in 10-100KDa, swimming lane 3 that focuses mostly on is reduced, and is precipitated in swimming lane 4
More foreign protein out, to remove a part of foreign protein.Lipoxygenase band significantly reduces in swimming lane 5, and in swimming lane 6 in addition to
Be settled out LOX, be also settled out least a portion of foreign protein, it is therefore desirable to be concentrated by 30KDa ultrafiltration membrane and gel filtration chromatography into
The purifying of one step.It can be seen from the figure that LOX extracting solution is in single protein band by being further purified, illustrate that this was purified
Journey has obtained the LOX of higher degree, to achieve the purpose that isolate and purify LOX in soya whey wastewater.
LOX purification result in 2 soya whey wastewater of table
By pH6.0 precipitating dissolution after through 30KDa ultrafiltration membrane be concentrated and SephadexG-75 gel chromatography after LOX it is final
LOX purity is 90.43%, and specific enzyme activity reaches 25546.08U/mg, and the enzyme activity rate of recovery is 40.29%, and purification improves 25.44
Times, and heteroproteins band in chromatographic solution can be seen that according to PAGE gel electrophoretic analysis result and significantly reduce, purifying effect
Fruit is more significant.Therefore, which can be used for the development and utilization of LOX in soya whey wastewater, to realize resource utilization
Utilize the target of bioactive substance in soya whey wastewater.
Claims (10)
1. a kind of method that soya whey wastewater extracts lipoxygenase against pH gradient, which is characterized in that comprising steps of
1) soya whey wastewater pre-processes
The impurity that soya whey wastewater is carried out to centrifugation removal bulky grain retains supernatant and pre-processes as soya whey wastewater
Liquid；
2) foreign protein in soya whey wastewater pretreatment fluid is removed
The pH of soya whey wastewater pretreatment fluid is adjusted to 7.8 ~ 8.2, is centrifuged off precipitating；
3) lipoxygenase is extracted
The pH value that step 2 is centrifuged resulting supernatant is adjusted to 5.7 ~ 6.0, centrifugation removal supernatant, gained sediment is steamed
Distilled water dissolution, as lipoxygenase crude enzyme liquid.
2. the method that soya whey wastewater as described in claim 1 extracts lipoxygenase against pH gradient, it is characterised in that: also wrap
Step 4) is included, lipoxygenase crude enzyme liquid utilizes the method preliminary purification being concentrated by ultrafiltration.
3. the method that soya whey wastewater as claimed in claim 2 extracts lipoxygenase against pH gradient, it is characterised in that: also wrap
Step 5) is included, enzyme solution obtained by step 4) is further purified using gel filtration chromatography, obtains pure lipoxygenase.
4. the method that soya whey wastewater as claimed in claim 2 extracts lipoxygenase against pH gradient, it is characterised in that: step
4) in, using the modified poly (ether-sulfone) ultrafiltration membrane of 30KDa size, lipoxygenase crude enzyme liquid is carried out at 200 ~ 300KPa of pressure
8 ~ 10 times of ultrafiltration concentrations.
5. the method that soya whey wastewater as claimed in claim 2 extracts lipoxygenase against pH gradient, it is characterised in that: step
4) it in, is concentrated by ultrafiltration in such a way that ultrafiltration system is using double-filtration.
6. the method that soya whey wastewater as claimed in claim 3 extracts lipoxygenase against pH gradient, which is characterized in that step
5) in, gel filtration chromatography utilizes the Sephadex G-75 gel column of 2-70KDa, with pH7.8, the phosphoric acid of 0.02mol/L
Buffer balances 2 ~ 3 column volumes, and loading after balance, with pH7.8, the phosphate buffer of 0.02mol/L is washed for eluent
It is de-；Eluent flow rate is 0.5mL/min.
7. the method that soya whey wastewater as described in claim 1 extracts lipoxygenase against pH gradient, it is characterised in that: step
2) in, the pH of soya whey wastewater pretreatment fluid is adjusted to 8.0.
8. the method that soya whey wastewater extracts lipoxygenase against pH gradient as described in claim 1 or 7, it is characterised in that:
In step 3), the pH value of supernatant is adjusted to 6.0.
9. the method that soya whey wastewater as described in claim 1 extracts lipoxygenase against pH gradient, it is characterised in that: step
2) and in step 3), the pH of sodium hydroxide solution regulation system is utilized.
10. the method that soya whey wastewater as described in claim 1 extracts lipoxygenase against pH gradient, it is characterised in that: step
It is rapid 1) in, 8 ~ 20min is centrifuged with the revolving speed of 3000 ~ 15000r/min.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112626041A (en) * | 2020-12-24 | 2021-04-09 | 齐鲁工业大学 | Process for extracting lipoxygenase from soybean whey protein wastewater by two aqueous phases |
| CN116693660A (en) * | 2023-08-08 | 2023-09-05 | 山东禹王生态食业有限公司 | Extraction method of soybean trypsin inhibitor |
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| US20100144009A1 (en) * | 2006-12-20 | 2010-06-10 | Protech Research Pty Ltd. | Extracting And Purifying Lipoxygenase |
| WO2014179207A2 (en) * | 2013-04-29 | 2014-11-06 | US NewWin, Inc. | Enhanced heterologous production of lipoxygenases |
| CN106858615A (en) * | 2017-03-08 | 2017-06-20 | 吉林大学 | One kind contains compound coarse food grain albumen powder of anti-oxidation peptide and preparation method thereof |
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2019
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| US20100144009A1 (en) * | 2006-12-20 | 2010-06-10 | Protech Research Pty Ltd. | Extracting And Purifying Lipoxygenase |
| WO2014179207A2 (en) * | 2013-04-29 | 2014-11-06 | US NewWin, Inc. | Enhanced heterologous production of lipoxygenases |
| CN106858615A (en) * | 2017-03-08 | 2017-06-20 | 吉林大学 | One kind contains compound coarse food grain albumen powder of anti-oxidation peptide and preparation method thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112626041A (en) * | 2020-12-24 | 2021-04-09 | 齐鲁工业大学 | Process for extracting lipoxygenase from soybean whey protein wastewater by two aqueous phases |
| CN116693660A (en) * | 2023-08-08 | 2023-09-05 | 山东禹王生态食业有限公司 | Extraction method of soybean trypsin inhibitor |
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